Bradley S. Launikonis

Store-operated Ca2+ entry during intracellular Ca2+ release in mammalian skeletal muscle.

Store‐operated Ca2+ entry (SOCE) is activated following the depletion of internal Ca2+ stores in virtually all eukaryotic cells. Shifted excitation and emission ratioing of fluorescence (SEER) was used to image mag‐indo‐1 trapped in the tubular (t) system of mechanically skinned rat skeletal muscle fibres to measure SOCE during intracellular Ca2+ release. Cytosolic Ca2+ transients were simultaneously imaged using the fluorescence of rhod‐2. Spatially and temporally resolved images of t system [Ca2+] ([Ca2+]t‐sys) allowed estimation of Ca2+ entry flux from the rate of decay of [Ca2+]t‐sys. Ca2+ release was induced pharmacologically to activate SOCE without voltage‐dependent contributions to Ca2+ flux. Inward Ca2+ flux was monotonically dependent on the [Ca2+] gradient, and strongly dependent on the transmembrane potential. The activation of SOCE was controlled locally. It could occur without full Ca2+ store depletion and in less than a second after initiation of store depletion. These results indicate that the molecular agonists of SOCE must be evenly distributed throughout the junctional membranes and can activate rapidly. Termination of SOCE required a net increase in [Ca2+]SR. Activation and termination of SOCE are also demonstrated, for the first time, during a single event of Ca2+ release. At the physiological [Ca2+]t‐sys, near 2 mm (relative to t system volume), SOCE flux relative to accessible cytoplasmic volume was at least 18.6 μm s−1, consistent with times of SR refilling of 1–2 min measured in intact muscle fibres.

Identification of the coupling between skeletal muscle store-operated Ca2+ entry and the inositol trisphosphate receptor

Examination of store-operated Ca2+ entry (SOC) in single, mechanically skinned skeletal muscle cells by confocal microscopy shows that the inositol 1,4,5-trisphosphate (IP3) receptor acts as a sarcoplasmic reticulum [Ca2+] sensor and mediates SOC by physical coupling without playing a key role in Ca2+ release from internal stores, as is the case with various cell types in which SOC was investigated previously. The results have broad implications for understanding the mechanism of SOC that is essential for cell function in general and muscle function in particular. Moreover, the study ascribes an important role to the IP3 receptors in skeletal muscle, the role of which with respect to Ca2+ homeostasis was ill defined until now.

Inhibitory control Over Ca2+ Sparks via Mechanosensitive Channels Is Disrupted in Dystrophin Deficient Muscle but Restored by Mini-Dystrophin Expression

Background In dystrophic skeletal muscle, osmotic stimuli somehow relieve inhibitory control of dihydropyridine receptors (DHPR) on spontaneous sarcoplasmic reticulum elementary Ca2+ release events (ECRE) in high Ca2+ external environments. Such ‘uncontrolled’ Ca2+ sparks were suggested to act as dystrophic signals. They may be related to mechanosensitive pathways but the mechanisms are elusive. Also, it is not known whether truncated dystrophins can correct the dystrophic disinhibition. Methodology/Principal Findings We recorded ECRE activity in single intact fibers from adult wt, mdx and mini-dystrophin expressing mice (MinD) under resting isotonic conditions and following hyper-/hypo-osmolar external shock using confocal microscopy and imaging techniques. Isotonic ECRE frequencies were small in wt and MinD fibers, but were markedly increased in mdx fibers. Osmotic challenge dramatically increased ECRE activity in mdx fibers. Sustained osmotic challenge induced marked exponential ECRE activity adaptation that was three times faster in mdx compared to wt and MinD fibers. Rising external Ca2+ concentrations amplified osmotic ECRE responses. The eliminated ECRE suppression in intact osmotically stressed mdx fibers was completely and reversibly resuscitated by streptomycine (200 µM), spider peptide GsMTx-4 (5 µM) and Gd3+ (20 µM) that block unspecific, specific cationic and Ca2+ selective mechanosensitive channels (MsC), respectively. ECRE morphology was not substantially altered by membrane stress. During hyperosmotic challenge, membrane potentials were polarised and a putative depolarisation through aberrant MsC negligible excluding direct activation of ECRE through tubular depolarisation. Conclusions/Significance Dystrophin suppresses spontaneous ECRE activity by control of mechanosensitive pathways which are suggested to interact with the inhibitory DHPR loop to the ryanodine receptor. MsC-related disinhibition prevails in dystrophic muscle and can be resuscitated by transgenic mini-dystrophin expression. Our results have important implications for the pathophysiology of DMD where abnormal MsC in dystrophic muscle confer disruption of microdomain Ca2+ homeostasis. MsC blockers should have considerable therapeutic potential if more muscle specific compounds can be found.

Ca2+ Sparks and Embers of Mammalian Muscle. Properties of the Sources

Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were “lone embers.” Average spatial width was ∼2 μm in the rat and 1.5 μm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37–160 channels opened in the spark. In simulations, 25–60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 μm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35–260 channels. Not infrequently, a few of those remain open and produce the trailing ember.

Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle.

1 Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2 Treatment with 10 μg ml−1 saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min−1. Saponin concentrations up to 150 μg ml−1 and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10–150 μg ml−1 saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse‐tubular system. 3 Treatment with saponin at concentrations up to 100 μg ml−1 for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min−1 in the presence of 150 μg ml−1 saponin. 4 The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 μM Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5 Treatment of skinned fibres of long sarcomere length (> 6 μM) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 μg ml−1 saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6 It is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species dependent manner, probably by increasing the Ca2+ loss through SR Ca2+ release channels.

Confocal imaging of [Ca2+] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence

Intracellular calcium signals regulate multiple cellular functions. They depend on release of Ca2+ from cellular stores into the cytosol, a process that appears to be tightly controlled by changes in [Ca2+] within the store. A method to image free [Ca2+] within cellular organelles was devised, which provided the first quantitative confocal images of [Ca2+] inside the sarcoplasmic reticulum (SR) of skeletal muscle. The method exploits, for greater sensitivity, the dual spectral shifts that some fluorescent dyes undergo upon binding Ca2+. It was implemented with mag‐indo‐1 trapped in the intracellular organelles of frog skeletal muscle and validated showing that it largely monitors [Ca2+] in a caffeine‐sensitive compartment with the structure of the SR cisternae. A tentative calibration in situ demonstrated an increase in the dyes dissociation constant, not unlike that observed for other dyes in cellular environments. This increase, together with other characteristics of the ratioing method, placed the half‐signal [Ca2+] near 1 mm, a value suitable for cellular stores. Demonstrated advantages of the technique include accuracy (that of a calibrated ratiometric method), dynamic range and sensitivity (from the combination of two spectral shifts), spatial and temporal resolution, and compatibility with a vast array of visible dyes to monitor diverse aspects of cellular function. SEER (shifted excitation and emission ratioing) also provides a [Ca2+]‐independent measure of dye concentration in the cell. Store and mitochondrial [Ca2+] ([Ca2+]SR and [Ca2+]mito) could be measured separately using the high spatial resolution of SEER. Evolution of [Ca2+]SR was followed upon changes in cytosolic [Ca2+] ([Ca2+]cyto). At [Ca2+]cyto= 100 nm, [Ca2+]mito remained near the lower limit of detection and [Ca2+]SR stabilized at values that were submillimolar according to our tentative calibration. Steady [Ca2+]SR was only slightly higher in 800 nm[Ca2+]cyto, and essentially did not decrease unless [Ca2+]cyto was reduced below 10 nm. While the increase of [Ca2+]SR was limited by loss through Ca2+ release channels, its decrease in low [Ca2+]cyto was largely dependent on leaks through the SR Ca2+ pump.

Upregulation of store-operated Ca2+ entry in dystrophic mdx mouse muscle

Store-operated Ca(2+) entry (SOCE) is an important mechanism in virtually all cells. In adult skeletal muscle, this mechanism is highly specialized for the rapid delivery of Ca(2+) from the transverse tubule into the junctional cleft during periods of depleting Ca(2+) release. In dystrophic muscle fibers, SOCE may be a source of Ca(2+) overload, leading to cell necrosis. However, this possibility is yet to be examined in an adult fiber during Ca(2+) release. To examine this, Ca(2+) in the tubular system and cytoplasm were simultaneously imaged during direct release of Ca(2+) from sarcoplasmic reticulum (SR) in skeletal muscle fibers from healthy (wild-type, WT) and dystrophic mdx mouse. The mdx fibers were found to have normal activation and deactivation properties of SOCE. However, a depression of the cytoplasmic Ca(2+) transient in mdx compared with WT fibers was observed, as was a shift in the SOCE activation and deactivation thresholds to higher SR Ca(2+) concentrations ([Ca(2+)](SR)). The shift in SOCE activation and deactivation thresholds was accompanied by an approximately threefold increase in STIM1 and Orai1 proteins in dystrophic muscle. While the mdx fibers can introduce more Ca(2+) into the fiber for an equivalent depletion of [Ca(2+)](SR) via SOCE, it remains unclear whether this is deleterious.

Toward the roles of store-operated Ca2+ entry in skeletal muscle

Store-operated Ca2+ entry (SOCE) has been found to be a rapidly activated robust mechanism in skeletal muscle fibres. It is conducted across the junctional membranes by stromal interacting molecule 1 (STIM1) and Orai1, which are housed in the sarcoplasmic reticulum (SR) and tubular (t-) system, respectively. These molecules that conduct SOCE appear evenly distributed throughout the SR and t-system of skeletal muscle, allowing for rapid and local control in response to depletions of Ca2+ from SR. The significant depletion of SR Ca2+ required to reach the activation threshold for SOCE could only be achieved during prolonged bouts of excitation–contraction coupling (EC coupling) in a healthy skeletal muscle fibre, meaning that this mechanism is not responsible for refilling the SR with Ca2+ during periods of fibre quiescence. While Ca2+ in SR remains below the activation threshold for SOCE, a low-amplitude persistent Ca2+ influx is provided to the junctional cleft. This article reviews the properties of SOCE in skeletal muscle and the proposed molecular mechanism, assesses its potential physiological roles during EC coupling, namely refilling the SR with Ca2+ and simple balancing of Ca2+ within the cell, and also proposes the possibility of SOCE as a potential regulator of t-system and SR membrane protein function.

Ultra-rapid activation and deactivation of store-operated Ca2+ entry in skeletal muscle

Skeletal muscle is highly specialized for the rapid delivery of Ca(2+) to the contractile apparatus during excitation-contraction coupling (EC coupling). Previous studies have shown the presence of a relatively fast-activated store-operated Ca(2+) entry (SOCE) mechanism (<1s) to be present in skeletal muscle, unlike the situation occurring in non-excitable cells. We simultaneously imaged [Ca(2+)] in the t-system and cytoplasm in mechanically skinned fibers during SR Ca(2+) release and observed both cell-wide Ca(2+) release and Ca(2+) waves. SOCE activation followed cell-wide Ca(2+) release from high sarcoplasmic reticulum (SR) [Ca(2+)] ([Ca(2+)](SR)) by seconds, consistent with depletion of [Ca(2+)](SR) to an absolute threshold for SOCE and an unformed SOCE complex at high [Ca(2+)](SR). Ca(2+) waves occurred at low [Ca(2+)](SR), close to the threshold for SOCE, minimizing the time between Ca(2+) release and Ca(2+) influx. Local activation of SOCE during Ca(2+) waves occurred in approximately 27ms following local initiation of SR depletion indicating a steep relationship between [Ca(2+)](SR) and SOCE activation. Most of this delay was due to slow release of Ca(2+) from SR, leaving only milliseconds at most for the activation of Ca(2+) entry following store depletion. SOCE was also observed to deactivate effectively instantly during store refilling at low [Ca(2+)](SR). These rapid kinetics of SOCE persisted as subsequent Ca(2+) waves propagated along the fiber. Thus we show for the first time millisecond activation and deactivation of SOCE during low amplitude [Ca(2+)](SR) oscillations at low [Ca(2+)](SR). To account for the observed Ca(2+) movements we propose the SOCE complex forms during the progressive depletion of [Ca(2+)](SR) prior to reaching the activation threshold of SOCE and this complex remains stable at low [Ca(2+)](SR).

Regulation of Ca2+ Sparks by Ca2+ and Mg2+ in Mammalian and Amphibian Muscle. An RyR Isoform-specific Role in Excitation–Contraction Coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 μM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4–40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channels activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10−7 in the frog or 10−8 in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.